Rice disease susceptibility gene Os10g0506100 and application of the same in regulating plant disease resistance

Abstract

The application discloses a rice disease gene Os10g0506100 and application of an encoded protein thereof in regulation of plant disease resistance. The nucleotide sequence of the rice disease gene Os10g0506100 is shown in SEQ ID NO:1, and the protein sequence encoded by the gene is shown in SEQ ID NO:2. The application knocks out the Os10g0506100 gene in rice through RISPR / Cas9 technology, finds that knockout of the gene can significantly improve the resistance of rice to rice blast, and provides an effective approach for rice breeding and improvement of disease-resistant varieties.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of the rice susceptibility gene Os10g0506100 and its encoded protein in regulating plant disease resistance. Background Technology

[0002] Rice blast is one of the most destructive fungal diseases in rice production. The race differentiation and rapid evolution of pathogen populations in the field often lead to the gradual degradation of race-specific resistance based on a single resistance gene (R) after its introduction. Compared with the "acquired complete resistance" conferred by the R gene, targeting the host susceptibility gene (S gene) to obtain "disabled resistance" has advantages such as race-specificity, relatively longer durability, and strong complementarity with existing resistance sources, thus becoming a key direction for improving the durability of rice blast resistance.

[0003] At the host-side molecular level, protein families containing heavy metal-binding domains (HMAs) play a central role in the rice-blast disease interaction. On one hand, some R receptors integrate HMA domains to mediate effector recognition; on the other hand, several small HMA proteins have been shown to participate in the susceptibility process, such as Pi21 (OsHIPP05), HIPP19, and HIPP43 in rice. However, the HMA family has numerous phylogenetic branches, exhibiting diverse structures and functions, with different branches potentially undertaking different biological functions. There remains a problem of insufficient identification of key susceptibility members. Therefore, in the host network interacting with multiple effectors of rice blast fungus, potential "hub" factors may have amplifying effects on pathogen infection and host susceptibility pathways, and their identification is crucial for obtaining stable and broad-spectrum disease resistance improvement pathways. Summary of the Invention

[0004] The primary objective of this invention is to overcome the shortcomings of existing technologies in the application of HMA-type disease susceptibility genes in the breeding of disease-resistant rice, and to provide a protein encoded by the rice disease susceptibility gene Os10g0506100.

[0005] Another object of the present invention is to provide the nucleotide sequence of the rice susceptibility gene Os10g0506100.

[0006] Another object of the present invention is to provide the application of the rice susceptibility gene Os10g0506100.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] The amino acid sequence of the protein encoded by the rice susceptibility gene Os10g0506100 is shown in SEQ ID NO:2.

[0009] The nucleotide sequence of the rice susceptibility gene Os10g0506100 is shown in SEQ ID NO:1.

[0010] The expression cassette, recombinant vector, or recombinant bacteria containing the rice susceptibility gene Os10g0506100.

[0011] The application of the protein encoded by the rice susceptibility gene Os10g0506100, the rice susceptibility gene Os10g0506100, and at least one of the following: expression cassette containing the rice susceptibility gene Os10g0506100 or targeting the gene, recombinant vector, or recombinant bacteria, in regulating plant disease resistance, cultivating transgenic rice, or gene-edited rice.

[0012] The regulation of plant disease resistance refers to improving the plant's resistance to rice blast.

[0013] The plant in question is rice; more specifically, rice varieties with weaker disease resistance, such as Huang Huazhan.

[0014] The regulation is achieved by silencing or knocking out the rice susceptibility gene Os10g0506100. For example, the rice susceptibility gene Os10g0506100 can be knocked out using CRISPR gene editing technology. The resulting Os10g0506100 gene knockout rice without the CRISPR vector sequence exhibits significant rice blast resistance.

[0015] The sequence of the sgRNA used to knock out the rice susceptibility gene Os10g0506100 is shown in SEQ ID NO:3.

[0016] Furthermore, the knockout of the rice susceptibility gene Os10g0506100 is achieved through any of the following methods:

[0017] (a) A T base is deleted at position 50 in one allele of the rice susceptibility gene Os10g0506100 as shown in SEQ ID NO.1, and an A base is inserted between positions 49 and 50 in another allele (heterozygous mutant).

[0018] (b) Insert a base T between positions 49 and 50 of the rice susceptibility gene Os10g0506100 as shown in SEQ ID NO.1 (homozygous mutant).

[0019] A mutant of the rice susceptibility gene Os10g0506100, which is any one of the following:

[0020] (a) A T base is deleted at position 50 in one allele of the rice susceptibility gene Os10g0506100 as shown in SEQ ID NO.1, and an A base is inserted between positions 49 and 50 in another allele (heterozygous mutant).

[0021] (b) Insert a base T between positions 49 and 50 of the rice susceptibility gene Os10g0506100 as shown in SEQ ID NO.1 (homozygous mutant).

[0022] Expression cassettes, recombinant vectors, or recombinant bacteria containing the Os10g0506100 mutant of the rice susceptibility gene.

[0023] The application of the rice susceptibility gene Os10g0506100 mutant, and at least one of the expression cassette, recombinant vector, or recombinant bacteria containing the rice susceptibility gene Os10g0506100 mutant in improving plant disease resistance, cultivating transgenic rice, or gene-edited rice.

[0024] The improvement of plant disease resistance refers to improving the plant's resistance to rice blast.

[0025] The plant in question is rice; more specifically, rice varieties with weaker disease resistance, such as Huang Huazhan.

[0026] This invention identified the HMA-type candidate susceptibility gene Os10g0506100 through AI-based protein interaction prediction, bimolecular fluorescence complementation, and immunoprecipitation experiments. Compared with known HMA susceptibility genes Pi21, HIPP19, and HIPP43, Os10g0506100 is located in a different phylogenetic branch of the HMA family, indicating its independence and differences in structural features and functional localization, thus representing a novel susceptibility gene resource. Furthermore, this invention evaluated the rice blast resistance of Huang Huazhan (HHZ), a rice model created using RISPR / Cas9 technology with this gene knocked out, and found that knocking out this gene significantly improves rice resistance to rice blast. Details are as follows:

[0027] (1) Screening of Os10g0506100 gene knockout sgRNA target sites by blastn short sequence alignment; CRISPR gene knockout vectors were constructed using the Golden Gate Assembly method.

[0028] (2) The rice variety Huanghuazhan was transformed by Agrobacterium-mediated genetic transformation of plant callus to obtain T0 generation plants, and T1 generation plants were obtained by self-pollination.

[0029] (3) Using Sanger sequencing and DSDecode software, the genotypes of the Os10g0506100 target site in T0 and T1 generation plants were identified, and the Os10g0506100 biallelic knockout mutant was obtained.

[0030] (4) By agarose gel electrophoresis, we can identify whether the CRISPR vector sequence is preserved in the T1 generation plants and screen for Os10g0506100 bicelestek mutants without CRISPR vector sequence.

[0031] (5) The rice blast resistance of the Os10g0506100 bis-allelic knockout mutant without CRISPR vector sequence was verified by using the rice leaf blast fungus spot inoculation method.

[0032] (6) The rice blast resistance of the Os10g0506100 bis-allelic knockout mutant of the CRISPR vector sequence was verified by spraying rice seedlings with rice blast fungus.

[0033] The evidence provided by this invention regarding the extensive association between the Os10g0506100 gene and various effectors of rice blast fungus suggests that it may occupy a "hub" position in the pathogen-host interaction network. This characteristic makes it a preferred target for elucidating key nodes in the susceptibility pathway and conducting molecular modification. Therefore, the functional analysis and application of Os10g0506100 not only helps to expand the molecular map of the HMA family in rice susceptibility but also provides new genetic resources and theoretical basis for constructing durable and usable breeding strategies for rice blast resistance.

[0034] The present invention has the following advantages and effects compared with the prior art:

[0035] (1) This invention provides a new susceptibility gene Os10g0506100 and its encoded protein, which expands the rice HMA susceptibility gene network.

[0036] (2) The Os10g0506100 gene provided by the present invention is a preferred gene for gene editing for rice blast resistance breeding. It can be obtained by knocking out the single gene Os10g0506100, or by knocking out two or more genes in combination with known susceptible genes such as Pi21, HIPP19, and HIPP43, or by replacing the HMA domain integrated in the Pikm-1 or RGA5 resistance genes.

[0037] (3) The Os10g0506100 gene provided by this invention is conserved in the genome of rice varieties, which provides a way to improve some high-yield and high-quality varieties but with weak disease resistance, such as Huang Huazhan. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the Os10g0506100 gene knockout sgRNA target site screening.

[0039] Figure 2 This is a diagram showing the genotype identification results of the Os10g0506100 gene knockout strain.

[0040] Figure 3 This is a gel electrophoresis result of the CRISPR vector sequence being isolated and removed from the progeny lines.

[0041] Figure 4 This is a diagram showing the incidence of rice blast (spot inoculation with rice blast fungus Guy11) in wild-type rice Huanghuazhan HHZ (WT) and the Os10g0506100 gene knockout line CRISPR-KO#129 with the CRISPR vector sequence removed.

[0042] Figure 5 This is a graph showing the incidence of rice blast (sprayed with rice blast fungus Guy11) in wild-type HHZ (WT) rice and the Os10g0506100 gene knockout line CRISPR-KO#129 with the CRISPR vector sequence removed; where A represents the incidence of the disease in wild-type and knockout plants; and B represents the statistical results of the average number of lesions in wild-type and knockout plants. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the instruments, reagents, materials, etc. involved in the embodiments are all conventional instruments, reagents, materials, etc., already existing in the prior art, and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods, detection methods, etc. involved in the following embodiments are all conventional experimental methods, detection methods, etc., already existing in the prior art.

[0044] Example 1: Screening of sgRNA target sites for Os10g0506100 gene knockout and construction of CRISPR gene knockout vector

[0045] 1.1 Screening of sgRNA target sites for Os10g0506100 gene knockout

[0046] In the genome of the rice variety Huanghuazhan, the Os10g0506100 gene is located on the negative strand of chromosome 10, with the code Chr10_HHZ_25693496.1729, and has two exons ( Figure 1The nucleotide sequence is shown in SEQ ID NO. 1, and the amino acid sequence encoding the protein is shown in SEQ ID NO. 2. The N20NGG sequence extracted from the positive and negative strands of the first exon is a potential sgRNA target site. Using the blastn software with the parameter -task blastn-short, homologous sequences of N20NGG in Huang Huazhan's genome were searched. N20NGG sequences with no homologous sequences, few homologous bases, or homologous bases that are not NGG or the first 12 bases were selected as sgRNA target sites. In Huang Huazhan's Os10g0506100 gene, the optimal editing site is GTGAACCGCCGAAGGAATCAGGG, where GGG is the PAM site and GTGAACCGCCGAAGGAATCA is the Protospacer (…). Figure 1 ), and based on this, an sgRNA sequence (SEQ ID NO.3) was designed. The relevant sequences are as follows:

[0047] The cDNA sequence of the Os10g0506100 gene (SEQ ID NO.1) (408bp):

[0048] ATGGCTCAGGTAAGCAAGTGCAACTCTTGTTTATGGTTCAATTCCCTGATTCCTTCGGCGGTTCACGCCTTCGTGTCGTCGGATGATAATTCTCAGCAGAAGGTGGTGCTGAGGGTTCCAACCATGACCGACGACAAGATTAAGCAGAAAGCCATTGAAGCCGTTGCAGACATCTACGGTATCGATTCCATAGCTGCAGATTTA AAGGACAATAAGATGACCATCATCGGTGAAATGGACACAGTGGCAATTGCAAAGAAGTTAAAGAAGATTGGGAAGATTGACATTGTGTCTGTTGGACCTGCAAAAGAAGAGAAGAAGGAAGAAAAGAAAGAGGAGAAGAAAGAAGAGAAGAAGGAAGAGAAGAAGGAGGAGAAGAAAGAGGAAAAGAAAGAAGAGAAGAAATGA;

[0049] The protein sequence encoded by Os10g0506100 (SEQ ID NO.2) (135 aa):

[0050] MAQVSKCNSCLWFNSLIPSAVHAFVSSDDNSQQKVVLRVPTMTDDKIKQKAIEAVADIYGIDSIAADLKDNKMTIIGEMDTVAIAKKLKKIGKIDIVSVGPAKEEKKEEKKEEKKEEKKEEKKEEKKEEKKEEKK*;

[0051] Os10g0506100 sgRNA (SEQ ID NO. 3) (103bp):

[0052] GTGAACCGCCGAAGGAATCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT.

[0053] 1.2 Construction of CRISPR gene knockout vector

[0054] (1) Dilute the forward primer sgRNA-oligo1 and the reverse primer sgRNA-oligo2 (Table 1) to 10 µM, take 10 µL of each and add them to a 1.5 mL centrifuge tube. Add 80 µL of ddH2O to 100 µL, mix well and heat in a water bath at 95 °C for 1 min. Then cool naturally at room temperature to anneal the double-stranded sgRNA guide sequence.

[0055] Table 1 Primers for Os10g0506100 gene knockout vector construction, target site genotype identification, and CRISPR vector sequence detection.

[0056]

[0057] (2) Using the Golden Gate Assembly method, sgRNAguide was ligated to plasmid pYLsgRNA-OsU6a (Gift from Yao-Guang Liu, Addgene plasmid #66194). The reaction system is shown in Table 2.

[0058] Table 2

[0059]

[0060] Reaction program: 10 PCR cycles (37℃, 5 min; 20℃, 5 min).

[0061] (3) Two rounds of PCR reaction:

[0062] ① First round of PCR: Each sgRNA expression cassette is divided into 2 PCR reactions, each with a reaction system of 25 µL: Take 0.8 μL of ligation product as template, and use UF and sgRNA-oligo2 primers (reaction 1, primers are shown in Table 1, reaction system is shown in Table 3), and sgRNA-oligo1 and gR-R (reaction 2, primers are shown in Table 1, reaction system is shown in Table 4) for amplification.

[0063] Table 3

[0064]

[0065] Table 4

[0066]

[0067] Reaction program: 98℃, 2 min; 25 PCR cycles (98℃, 20 s; 60℃, 20 s; 72℃, 25 s).

[0068] ② Second round PCR: Based on the number of sgRNA expression cassettes and their intended placement in the vector, synthesize the corresponding specific amplification primers Position1-F and Position1-R (Table 1). Take 1 µL each of the products from reaction 1 and reaction 2 in the first round PCR and add them to 8 µL of ddH2O for a 10-fold dilution. Use 2 µL of this solution as the template for the second round PCR (mixed template). The reaction system is shown in Table 5.

[0069] Table 5

[0070]

[0071] Reaction program: 98℃, 2 min; 20 PCR instrument cycles (95℃, 20 s; 58℃, 20 s; 72℃, 25 s).

[0072] ③ The second round of PCR products were purified to obtain the OsU6a-sgRNA expression cassette.

[0073] (4) The OsU6a-sgRNA expression cassette was cloned into the pYLCRISPR / Cas9Pubi-H vector (Gift from Yao-Guang Liu, Addgene plasmid #66187) by the golden gate assembly method. The reaction system is shown in Table 6.

[0074] Table 6

[0075]

[0076] Reaction program: 10-15 PCR cycles (37℃, 5 min; 10℃, 5 min; 20℃, 5 min); 37℃, 5 min.

[0077] (5) Transform the ligation product into competent Escherichia coli cells DH5α, incubate at 37℃ for 12-16 h, then pick single colonies for colony PCR identification, select the corresponding bacterial solutions with correct electrophoretic bands and send them to the company for sequencing, and if the sequencing results are correct, preserve the bacteria and extract the plasmid to obtain the CRISPR knockout vector plasmid.

[0078] 2. Agrobacterium-mediated genetic transformation of rice

[0079] (1) Agrobacterium-mediated transformation of CRISPR knockout vector

[0080] Remove the 0.2 cm electroporation cuvette from the storage solution, drain off excess water, and place it on ice. Remove EHA105 Agrobacterium competent cells from the -80°C freezer and insert them into ice. After thawing, add 1 μL of the prepared CRISPR knockout vector plasmid, gently mix by hand, and incubate on ice for 2 min. Transfer the competent cells to the 0.2 cm electroporation cuvette using a pipette, place it in the electroporation tank, start the electroporator, set the parameters, and after electroporation, quickly add 1 ml of antibiotic-free YEP liquid medium. Incubate at 28°C for 2 hours on a shaker at 200 rpm. Dilute the bacterial culture 10-fold and spread it on a YEP plate containing Rif (25 µg / mL) + Kan (50 µg / mL). After single colonies grow and are correctly identified, preserve the bacterial strain.

[0081] (2) Genetic transformation of rice

[0082] a. Select plump, mold-free Huanghuazhan rice seeds, wash them with 75% ethanol for 90 seconds, discard the ethanol, add 2.5% sodium hypochlorite, place them in a shaker and shake at room temperature for 30 minutes, discard the sodium hypochlorite, wash them 5-10 times with ddH2O, blot dry on filter paper, and then inoculate the rice seeds into an induction medium (3.98 g / L Chu (N6) vitamin-free basal salt (PhytoTech Labs), 1 ml modified vitamin (1000×) (PhytoTech Labs), 3 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 g / L casein peptone, 2.5 g / L proline, 30 g / L sucrose, 3 g / L plant gel (PhytoTech Labs), pH 5.8), and culture at 26℃ under light for 20 days to induce callus tissue.

[0083] b. Pick a single Agrobacterium colony and inoculate it in an infection solution [1× AB salt (2 g / L ammonium chloride, 0.3 g / L magnesium sulfate, 0.15 g / L potassium chloride, 0.01 g / L calcium chloride, 2.5 mg / L ferrous sulfate), 2 mM phosphate, 20 mM MES (2-(N-morpholine) ethanesulfonic acid (PytoTech Labs), 1% (w / v) glucose, 200 mM acetylsuccinone, 50 μg / mL kanamycin, pH 5.5). After culturing for 24 hours, collect the cells; prepare an Agrobacterium resuspension with OD600 = 0.2 (3.98 g / L Chu(N6) vitamin-free basal salt, 1 ml modified vitamin (1000×), 0.5 g / L casein peptone, 30 g / L sucrose, 10 g / L glucose, 100 mg / L ferrous sulfate, 2 mM phosphate, 20 mM MES (2-(N-morpholine) ethanesulfonic acid (PytoTech Labs), 1% (w / v) glucose, 200 mM acetylsuccinate, 50 μg / mL kanamycin, pH 5.5). µM acetylsuccinone (pH 5.5), use sterile forceps to place the callus tissue into an Erlenmeyer flask containing Agrobacterium resuspension, shake for 10-15 min and then discard the bacterial solution;

[0084] c. Inoculate the callus onto a co-culture medium (3.98 g / L Chu (N6) vitamin-free basal salt, 1 ml modified vitamin (1000×), 0.5 g / L casein peptone, 2 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 30 g / L sucrose, 10 g / L glucose, 100 μM acetylsalicylic acid, 3 g / L plant gel, pH 5.5) and co-culture at 20℃ for 48~72 h;

[0085] d. Using sterile forceps, pick up callus tissue and inoculate it into selection medium (3.98 g / L Chu (N6) vitamin-free basal salts, 1 ml modified vitamin (1000×), 2 mg / L 2,4-D, 0.3 g / L caseinate hydrolysate, 2.88 g / L proline, 30 g / L maltose, 400 mg / L termethin, 50 mg / L hygromycin, 3 g / L plant gel, pH 5.8), and incubate in the dark at 26℃ for 20-30 days; pick up positive monoclonal callus tissue and transplant the callus tissue back into selection medium, and incubate in the dark at 26℃ for 7-10 days;

[0086] e. Inoculate hygromycin-resistant callus into differentiation medium (3.98 g / L Chu (N6) vitamin-free basal salt, 1 ml modified vitamin (1000×), 2 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.3 g / L casein peptone, 2.88 g / L proline, 30 g / L maltose, 400 mg / L termethin, 50 mg / L hygromycin, 3 g / L plant gel, pH 5.8) and culture at 25-27℃ for 15-20 days until shoots of 2-5 cm in length differentiate.

[0087] f. Subsequently, these buds were transferred to rooting medium (4.3 g / L MS basal salt (PhytoTech Labs), 1 ml modified vitamin (1000×), 30 g / L sucrose, 50 mg / L hygromycin, 3 g / L Gelrite (PhytoTech Labs), pH 5.8) and cultured for 7-10 days under 30°C light to promote root growth and obtain regenerated seedlings.

[0088] 3. Genotyping of Os10g0506100 gene knockout plants

[0089] (1) Extraction of rice genomic DNA

[0090] DNA from gene-edited rice plants was extracted using the CTAB method, as follows: A prepared CTAB (hexadecyltrimethylammonium bromide) solution was preheated to 65 °C in a water bath. A suitable amount of young rice leaf tissue was quickly placed in a mortar and ground into powder using liquid nitrogen. The ground sample was transferred to a 1.5 mL centrifuge tube using a spatula, and 500 μL of preheated CTAB solution (65 °C) was quickly added and vortexed to mix. The centrifuge tube was then placed in a 65 °C water bath for 30 min, inverting and mixing every 10 min. After the sample cooled, an equal volume of chloroform-isoamyl alcohol mixture (24:1) was added, and the mixture was inverted and mixed. The mixture was then centrifuged at 12000 rpm for 3-5 min. The supernatant was transferred to a new 1.5 mL centrifuge tube, and an equal volume of isopropanol was added. The tube was placed in a -20 °C freezer for 30 min, and then centrifuged at 12000 rpm for 10 min. Remove the supernatant with a pipette, add 1 mL of 70% ethanol to wash the precipitate, and repeat this step twice. Aspirate the ethanol from the precipitate, retaining the precipitate. Open the 1.5 mL centrifuge tube and allow it to air dry to remove any residual ethanol. Dissolve the precipitate in 30–50 μL of ddH₂O and store at -20°C.

[0091] (2) Genotyping of T1 generation rice plants

[0092] Genomic DNA extracted from T0 or T1 generation lines was amplified using primers Os10g0506100-F and Os10g0506100-R (Table 1). The PCR reaction system is shown in Table 7.

[0093] Table 7

[0094]

[0095] Reaction program: 98℃, 2 min; 25 PCR cycles (98℃, 10 s; 60℃, 10 s; 72℃, 8 s); 72℃, 5 min.

[0096] PCR products were sent to the company for Sanger sequencing. Sequencing data were analyzed using DSDecode (http: / / www.crispr-ge.com / superdecode / dsdecode / ) to obtain the Os10g0506100 knockout genotype. A total of four Os10g0506100 biallelic mutant lines were screened, namely CRISPR-KO#118, 129, 130, and 132. Figure 2 Among them, the CRISPR-KO#118 strain is a heterozygous mutant, meaning that one allele has an insertion of a nucleotide (a T base is missing at position 50 of SEQ ID NO.1), and the other allele has a missing nucleotide (an A base is inserted between positions 49 and 50 of SEQ ID NO.1). In the CRISPR-KO#129, #130, and #132 strains, the Os10g0506100 gene is a homozygous mutant, meaning that both alleles have an insertion of a nucleotide (a T base is inserted between positions 49 and 50 of SEQ ID NO.1).

[0097] 4. T1 generation CRISPR vector sequence detection

[0098] Genomic DNA of the T1 generation line was amplified using primers Cas9-F and Cas9-R (Table 1). The PCR reaction system is shown in Table 8.

[0099] Table 8

[0100]

[0101] Reaction program: 98℃, 2 min; 25 PCR cycles (98℃, 10 s; 60℃, 10 s; 72℃, 9 s); 72℃, 5 min.

[0102] After gel electrophoresis of the amplified products, the retention status of the CRISPR vector sequence in the T1 line was obtained. Figure 3 () Figure 3 In the CRISPR-KO series, #115 to #132 represent the seed numbers of T1 at the time of sowing. In the four lines CRISPR-KO#118, 129, 130, and 132, the CRISPR vector sequence was removed through segregation during the self-pollination process of the T0 line to generate the T1 line. This is combined with the genotype of the Os10g0506100 gene knockout in the T1 line (…). Figure 2The CRISPR-KO#118, 129, 130, and 132 lines are biallelic knockout mutants without the CRISPR vector sequence.

[0103] 5. Inoculation of Os10g0506100 gene knockout lines with rice blast fungus

[0104] (1) In the culture room, sow about 15 seeds per pot of Huanghuazhan wild type (HHZ (WT)) and Os10g0506100 gene knockout line CRISPR-KO#118, 129, 130, 132 T1 generation seeds. Culture for 4-5 weeks at 28℃ and 14 hours light. During this period, top-dress with 5 g / L ammonium sulfate (NH4SO2) solution at least twice. Take rice leaves for later use.

[0105] (2) Rice blast fungus strains Guy11, CHL346, and CHL508(FJ) were cultured on oat medium [Note: Guy11 is a commonly used rice blast fungus strain in this field and can be obtained through conventional methods; CHL346 and CHL508(FJ) were strains collected and identified from rice fields and are backed up and stored at the Agricultural Biotechnology Research Center of Guangdong Academy of Agricultural Sciences], and cultured under full light in a 368 nm black light incubator at a distance of about 25 cm, at 25-26℃ for about 14 days. When the mycelium basically covered the medium and turned gray, 0.25% (w / v) sterile gelatin solution was added to a clean bench, and the conidia were washed off by gently scraping with a spreader. The spore suspension was aspirated with a pipette, filtered through double-layer medical gauze, and collected in a sterile 50 mL centrifuge tube. The eluted and filtered conidial suspension was examined under a microscope, and the spore count was determined using a hemocytometer. The spore concentration was then adjusted to a final concentration of 1 × 10⁻⁶. 5 ~5×10 5 Add spores / mL to Tween-20 to the spore suspension to achieve a final concentration of 0.02% (v / v) to increase the adhesiveness of the spore suspension. Place on ice for later use.

[0106] (3) Prepare culture dishes for rice leaves to be inoculated, with 2-3 layers of circular filter paper about 9 cm in diameter laid flat, and the filter paper moistened with purified water. Select gene knockout plants and wild-type Huanghuazhan plants, cut 3 leaf segments about 5 cm long from each, place them flat in the prepared culture dishes, and use a pipette to drop 10 μL of rice blast fungus conidia suspension into the center of the leaf. Incubate at 25℃ for 16 hours under light. After 7 days of culture, photograph and record the disease situation. Inoculate Huanghuazhan and 4 gene knockout lines CRISPR-KO#118, 129, 130, 132 with 3 strains Guy11, CHL346 and CHL508(FJ), respectively, and obtain similar results. Figure 4 Leaves of Guy11 plants inoculated with Huang Huazhan and CRISPR-KO#129 were photographed for observation. On the leaves of the wild-type Huang Huazhan plants, clear brown lesions were visible at the inoculation site of the rice blast fungus, and these lesions expanded and converged towards the leaf margins. In contrast, no obvious brown lesions were observed on the CRISPR-KO lines.

[0107] (4) Cut rice leaves 48 hours after inoculation with rice blast fungus, place the leaves in a 2 mL centrifuge tube, and fix with 100% ethanol for 2-5 days to bleach the leaves and completely remove chlorophyll. After bleaching, replace the ethanol with 10% (w / v) KOH solution and heat in a water bath at 85℃ for 5 min to soften the leaves. Wash the sample 4-5 times with PBS solution (pH 7.4) to neutralize. Add the staining solution (PBS solution containing 10 μg / mL WGA-AF488 and 20 μg / mL PI) to the centrifuge tube, ensuring the sample is completely immersed in the staining solution. Vacuum the sample 3-4 times, 5 min each time, with a 5 min interval. Wash the sample twice with PBS (pH 7.4), then transfer it to a fresh PBS solution and store it at 4℃ in the dark for later use. Observe the growth of rice blast fungus hyphae in the leaf tissue using a confocal microscope. Three bacterial strains, Guy11, CHL346, and CHL508(FJ), were inoculated into Huang Huazhan and four gene knockout lines, CRISPR-KO#118, 129, 130, and 132, respectively, with three replicates yielding similar results. Figure 4 Confocal microscopy observation of leaves after Guy11 was inoculated with Huang Huazhan and CRISPR-KO#129: 48 hours after inoculation, in the wild-type strain Huang Huazhan, the hyphae of rice blast fungus filled the epidermal cells of the rice leaves it infected, while in the epidermal cells of CRISPR-KO plants, there were only a few hyphae, indicating that the infection of rice blast fungus was inhibited in CRISPR-KO plants.

[0108] 6. Spray inoculation of Os10g0506100 gene knockout line with rice blast fungus

[0109] Rice blast fungus strains were cultured using the same method described above, as well as wild-type HHZ (WT) and Os10g0506100 gene knockout CRISPR-KO plants of rice. Rice seedlings approximately two weeks old were used, and a concentration of 1.5 × 10⁻⁶ was applied. 5Spores of the rice blast fungus strain at a concentration of spores / L were evenly sprayed onto the upper and lower surfaces of rice leaves. After inoculation, rice seedlings were placed in an incubator at 22℃ and 100% humidity for 24 hours in the dark. After air drying at room temperature, they were then placed in a culture room at 28℃ with 16 hours of light. About one week after inoculation, the susceptibility of rice plants was recorded and analyzed. Six leaf segments of 10cm length from corresponding parts of each strain were taken, and the number of lesions was counted. A negative binomial regression model was used for statistical analysis of the number of lesions. Three strains, Guy11, CHL346, and CHL508(FJ), were inoculated into Huang Huazhan and four gene knockout lines, CRISPR-KO#118, 129, 130, and 132, respectively, with three replicates, yielding similar results. Figure 5 Observation and statistical analysis of disease incidence in Guy11 after inoculation with Huang Huazhan and CRISPR-KO#129: The average number of lesions in the Huang Huazhan strain was approximately 52.8 (95% confidence interval: 47.23-58.86), while the average number of lesions in the CRISPR-KO strain was 12.2 (95% confidence interval: 9.67-15.3). Compared with the wild-type Huang Huazhan, the number of lesions in the CRISPR-KO strain decreased by approximately 77%, a statistically significant difference (p < 2e). -16 ).

[0110] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of the protein encoded by the rice susceptibility gene Os10g0506100 or the rice susceptibility gene Os10g0506100 in regulating plant disease resistance, characterized by: The protein sequence encoded by the rice susceptibility gene Os10g0506100 is shown in SEQ ID NO:2; The nucleotide sequence of the rice susceptibility gene Os10g0506100 is shown in SEQ ID NO:1; The regulation of plant disease resistance mentioned above refers to improving the resistance of rice to leaf blast disease; The aforementioned regulation is achieved by knocking out the rice susceptibility gene Os10g0506100; The knockout of the rice susceptibility gene Os10g0506100 is achieved through any of the following methods: (a) In the rice susceptibility gene Os10g0506100, one allele is missing a nucleotide, namely, a T base is missing at position 50 of SEQ ID NO.1, and the other allele is inserted with a nucleotide, namely, an A base is inserted between positions 49 and 50 of SEQ ID NO.1; (b) Insert a T base between positions 49 and 50 of the rice susceptibility gene Os10g0506100 as shown in SEQ ID NO.

1.

2. The application of the protein encoded by the rice susceptibility gene Os10g0506100 or the rice susceptibility gene Os10g0506100 in gene-edited rice, characterized by: The protein sequence encoded by the rice susceptibility gene Os10g0506100 is shown in SEQ ID NO:2; The nucleotide sequence of the rice susceptibility gene Os10g0506100 is shown in SEQ ID NO:1; The gene-edited rice described is designed to improve rice's resistance to leaf blast disease. The gene-edited rice was achieved by knocking out the rice disease-susceptibility gene Os10g0506100; The knockout of the rice susceptibility gene Os10g0506100 is achieved through any of the following methods: (a) In the rice susceptibility gene Os10g0506100, one allele is missing a nucleotide, namely, a T base is missing at position 50 of SEQ ID NO.1, and the other allele is inserted with a nucleotide, namely, an A base is inserted between positions 49 and 50 of SEQ ID NO.1; (b) Insert a T base between positions 49 and 50 of the rice susceptibility gene Os10g0506100 as shown in SEQ ID NO.1.

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